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Title: Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even ifmore » their epitopes overlap.« less
 [1] ; ORCiD logo [2] ;  [2] ;  [2] ;  [3] ; ORCiD logo [4] ; ORCiD logo [2]
  1. Univ. of Zurich (Switzerland). Dept. of Biochemistry; Genentech, San Francisco, CA (United States). Dept. of Early Discovery Biochemistry
  2. Univ. of Zurich (Switzerland). Dept. of Biochemistry
  3. Univ. of Zurich (Switzerland). Dept. of Biochemistry; Federal Inst. of Technology, Zurich (Switzerland). Dept. of Biosystems Science and Engineering
  4. Univ. of Zurich (Switzerland). Dept. of Biochemistry; SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
Publication Date:
Grant/Contract Number:
AC02-76SF00515; 310030B_166676
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2045-2322
Nature Publishing Group
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE; Schweizerische Nationalfonds
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Fluorescent proteins; Protein design; Protein folding; X-ray crystallography
OSTI Identifier: